SANTOS, A. A. Q. A. Papel do receptor P2x7 no efeito da toxina a do Clostridium difficile em modelo de alça ileal em camundongos. 2017. 100 f. Tese (Doutorado em Ciências Médicas) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-30T16:00:58Z No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) / Approved for entry into archive by Erika Fernandes (erikaleitefernandes@gmail.com) on 2017-01-30T16:01:09Z (GMT) No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) / Made available in DSpace on 2017-01-30T16:01:09Z (GMT). No. of bitstreams: 1 2017_tese_aaqasantos.pdf: 2658637 bytes, checksum: 8c134a5e88286c7d898fc1d1a091d893 (MD5) Previous issue date: 2017-01-16 / Clostridium difficile (C. difficile) is the major cause of colitis associated with the use of antibiotics, with significant morbidity and mortality. The enteric nervous system (ENS) is located in the gastrointestinal tract (GIT) and has an important function of regulating the digestive system. The P2X7 receptor participates in the regulation of cellular permeability, cytokine release and apoptosis, processes that are involved in the pathogenesis of C. difficile induced disease.The aim of this study was to analyze the role of the P2X7 receptor in enteritis induced by C. difficile toxin A (TcdA). The mouse ileal loop was injected with saline (Sodium Chloride 0.9% - Control) or TcdA (50 μg / loop) in mice previously treated with the inhibitors: BBG (50 mg/kg) or A438079 (10 μM) i.p.injection 1 h prior to TcdA (TcdA + BBG; TcdA + A438079) or saline injection. The animals were euthanized after 4h latter and the ileal loop collected for the following analyzes: immunofluorescence for ChAT, NOS, P2X7 and calretinin in membrane preparations, immunohistochemistry for GFAP, HuC / D, S-100β, TUNEL and PCR for P2X7. The results demonstrated that TcdA significantly reduced myenteric plexus neurons in 66.42% when compared to the control, while in the membrane preparations a significant reduction of the immunoreactive neurons to ChAT was observed in 23.21% and Calretinin 28.17%. However, there was a 21.23% increase in labeling for the P2X7 receptor over the control. In the ileal tissue there was an increase of 411.95% and 22.40% respectively in the P2X7 positive area and in the gene expression. In relation to the glial markers, an increase of 125.71% and 144.30% was observed for GFAP and S-100β, respectively. In animals treated with inhibitors P2X7; BBG and A438079, as observed an improvement in histological parameters . In addition , a significant reduction in the number of apoptotic cells decrease in GFAP and S-100β-labeled cells, and cytokines ( TNF-α, IL-1β, IL -6 and IL-8) levels, compared to the control. This was associated with a significant increase in the HuC/D positive area . From these results, it is concluded that TcdA promotes changes in the enteric nervous system, increasing neuronal death and activating glial cells, and that the P2X7 receptor plays an important role in the changes induced by C. difficile TcdA. / O Clostridium difficile (C. difficile) é a maior causa de colite associada ao uso de antibióticos, com significante morbidade e mortalidade. O sistema nervoso entérico (SNE) está localizado no trato gastrointestinal (TGI) e possui importante função de regulação do sistema digestório. O receptor P2X7 presente nas células gliais entéricas (CGE) e no SNE participa na regulação da permeabilidade celular, liberação de citocinas e apoptose, processos que estam envolvidos na patogênese do C. difficile. O objetivo deste estudo foi analisar o papel do receptor P2X7 na enterite induzida pela toxina A (TcdA) do C. difficile. A alça ileal dos camundongos foi injetada com salina (Cloreto de Sódio 0,9%- Controle) ou TcdA (50 μg/alça) em camundongos previamente tratados com os inibidores: BBG (50 mg/kg) ou A438079 (10 μM) via intraperitoneal 1h antes da injeção da TcdA ou salina. Os animais foram eutanaziados após 4h da injeção e a alça ileal coletada para as seguintes analises: imunofluorescência para ChAT, NOS, P2X7 e calretinina nos preparados de membrana, imunohistoquímica para GFAP, HuC/D, S-100β, TUNEL e PCR para P2X7. Os resultados demonstraram que a TcdA reduziu significativamente em 66,42% os neurônios do plexo mioentérico quando comparado ao controle, enquanto nos preparados de membrana observou-se uma redução significativa dos neurônios imunorreativos a ChAT em 23,21% e Calretinina 28,17%, contudo, houve um aumento de 21,23% da marcação para o receptor P2X7 em relação ao controle. No tecido ileal verificou-se um aumento de 411,95% e 22,40% respectivamente na área positiva para P2X7 e na expressão gênica. Em relação aos marcadores gliais, observou-se um aumento de 125,71% e 144,30% para GFAP e S-100β, respectivamente. Nos animais tratados com os inibidores de P2X7: BBG e o A438079 observou-se uma melhora dos parâmetros histológicos, uma redução significativa do número de células apoptóticas, das células marcadas para GFAP e S-100β, das citocinas: TNF-α, IL-1β, IL-6 e IL-8 com relação ao controle e um aumento significativo na área positiva para HuC/D em relação a TcdA. A partir desses resultados, concluiu-se que a TcdA promoveu alterações no sistema nervoso entérico, aumentando a morte neuronal e ativando as células da glia, e que o receptor P2X7 tem participação importante nas alterações induzidas pela TcdA do C. difficile.

O sistema purinérgico tem sido amplamente implicado em condições médicas, entre elas, o transtorno bipolar (TB). Até onde sabemos, nenhum estudo foi realizado com o objetivo de esclarecer a possível contribuição do receptor P2X7 (P2X7R) na patofisiologia do TB ou mesmo com o objetivo de revisar o que já fora previamente publicado sobre este assunto. Nos presentes trabalhos, nosso objetivo foi em primeiro lugar explorar a relação entre o receptor e esse transtorno e identificar os mecanismos pelos quais o P2X7R desempenha um papel no TB e por fim revisar a literatura, focando na sinalização purinérgica no TB, com ênfase na sinalização por ATP e adenosina, destacando o potencial papel do P2X7R na modulação da inflamação e ativação microglial em pacientes bipolares. Para o primeiro objetivo, analisamos o efeito de moduladores do P2X7R (BzATP, BBG e A438079) sobre o comportamento (atividade locomotora) e sobre marcadores de neuroinflamação (IL-1 , TNF- e IL-6), excitotoxicidade (GFAP, TBARS) e neuroplasticidade (BDNF) em um modelo farmacológico de mania induzido pelo tratamento agudo e crônico com D – anfetamina (AMPH) (2mg/kg). Demonstramos na atividade locomotora uma aparente falta de capacidade de resposta à AMPH por animais com P2X7R bloqueado ou ausente (P2X7R-/-). Da mesma forma, observamos que o P2X7R participa do aumento do ambiente próinflamatório e excitotóxico induzido pela AMPH, demonstrado pelo papel de reversão do bloqueio do P2X7R ou pela perda de resposta dos animais P2X7R-/-. Em conclusão, nossos resultados suportam a hipótese de que o P2X7R possui um papel na patofisiologia do TB, como sugerido pelo nosso modelo animal, principalmente por mediar a neuroinflamação e a excitotoxicidade e finalmente levando a mudanças comportamentais. Assim, acreditamos que o P2X7R possui potencial para se tornar um alvo terapêutico do TB. Baseado neste cenário, estudos mais detalhados do papel do P2X7R no TB são necessários, especialmente devido à sua capacidade de agir sobre a microglia e modular a neuroinflamação e a excitotoxicidade. / The purinergic system has been increasingly implicated in medical conditions, among them bipolar disorder (BD). Up to date, no study has been conducted in order to clarify the possible contribution of the P2X7 receptor (P2X7R) to BD pathophysiology or even to review the previously data about this subject. In the presents works, we aim firstly to explore the association between the receptor and the disorder and identify the mechanisms by which the P2X7R plays a role in BD and lastly to review the literature focused on purinergic signaling in BD, with an emphasis on ATP and adenosine signaling, highlighting the potential role of P2X7R in modulating inflammation and microglia activation in bipolar patients. For the first aim, we analyzed the P2X7R modulatory effect (BzATP, BBG and A438079) on behavior (locomotor activity) and on markers of neuroinflammation (IL-1 , TNF- and IL-6), excitotoxicity (GFAP, TBARS), and neuroplasticity (BDNF) in a pharmacological model of mania induced by acute and chronic D-amphetamine (AMPH) treatment (2 mg/kg). We demonstrate in the locomotor activity an apparent lack of responsiveness to AMPH by animals with blocked or absent P2X7R (P2X7R-/-). Likewise, we observed that the P2X7R participates in the AMPH-induced increase of proinflammatory and excitotoxicity environment, demonstrated by the reversal role of P2X7R blockage or the lack of response by P2X7R-/- mice. In conclusion, our results provide support for the hypothesis that P2X7R plays a role in BD pathophysiology, as suggested by our animal model, mainly by mediating neuroinflammation and excitotoxicity and ultimately leading to behavioral changes. Thus, we believe that P2X7R has the potential to become a therapeutic target of BD. Based on this scenario, a more detailed study of the role of P2X7R in BD is warranted, especially due to its ability to act on microglia and modulate neuroinflammation and excitotoxicity.

Submitted by André Chamelete null (andre_ecco@hotmail.com) on 2016-02-12T14:02:00Z No. of bitstreams: 1 Dissertação de Mestrado - FINAL.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-12T17:20:57Z (GMT) No. of bitstreams: 1 chamelete_a_me_sjrp.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) / Made available in DSpace on 2016-02-12T17:20:57Z (GMT). No. of bitstreams: 1 chamelete_a_me_sjrp.pdf: 663322 bytes, checksum: 5ca648d67a3a798d08f9c68653ac3ca2 (MD5) Previous issue date: 2016-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O BaP é um contaminante ambiental capaz de causar inflamação e desregulação de vias celulares. Pela ação da CYP1a1 e CYP1b1, é convertido a metabólitos mais reativos. A literatura mostra que o BaP aumenta a expressão de algumas citocinas próinflamatórias, como a IL-1, porém, são bem contraditórios os relatos sobre o efeito do BaP no cFOS, o qual apresenta papel importante na proliferação, na formação de tumores e, possivelmente, na inflamação. O objetivo deste estudo foi de elucidar a participação do receptor purinérgico P2X7 sobre a expressão dos genes IL-1 e cFOS, durante exposição ao BaP. Foi empregado as técnicas de qPCR para quantificação de expressão gênica, e testes de correlação e regressão entre IL-1 e cFOS. A exposição ao BaP induziu a expressão dos dois genes, além das enzimas do seu metabolismo. Quando bloqueado o receptor P2X7, além de uma menor indução das CYPs, os níveis de IL-1 e cFOS caíram abaixo dos níveis controle, sugerindo a participação do P2X7. Os testes de correlação e regressão mostraram uma relação forte direta entre IL-1 e cFOS, reforçando o papel do cFOS na inflamação. / BaP is an environmental contaminant capable to cause inflammation and impair cellular pathways. CYP1a1 and CYP1b1 convert it to more reactive metabolites. Studies show that BaP enhances some proinflammatory citokines expression, like IL-1, yet reports about BaP affecting cFOS, which plays important role in proliferation, tumor formation and inflammation, are controversial. This work aimed to elucidate whether P2X7 purinergic receptor plays a role in IL-1 and cFOS expression during BaP exposure. We applied qPCR techniques to quantify gene expression, correlation and regression assays. Our results showed that BaP raised both IL-1 and cFOS genes expression, besides CYPs ones. Morevoer, when blocking P2X7 receptor, IL-1 and cFOS expression dropped under normal levels, which suggest P2X7 participation, in addition to a smaller enzymes induction. Correlation and regression assays exhibited a strong straight relationship between IL-1 and cFOS expression, reinforcing the role of cFOS in inflammation.

O sistema purinérgico tem sido amplamente implicado em condições médicas, entre elas, o transtorno bipolar (TB). Até onde sabemos, nenhum estudo foi realizado com o objetivo de esclarecer a possível contribuição do receptor P2X7 (P2X7R) na patofisiologia do TB ou mesmo com o objetivo de revisar o que já fora previamente publicado sobre este assunto. Nos presentes trabalhos, nosso objetivo foi em primeiro lugar explorar a relação entre o receptor e esse transtorno e identificar os mecanismos pelos quais o P2X7R desempenha um papel no TB e por fim revisar a literatura, focando na sinalização purinérgica no TB, com ênfase na sinalização por ATP e adenosina, destacando o potencial papel do P2X7R na modulação da inflamação e ativação microglial em pacientes bipolares. Para o primeiro objetivo, analisamos o efeito de moduladores do P2X7R (BzATP, BBG e A438079) sobre o comportamento (atividade locomotora) e sobre marcadores de neuroinflamação (IL-1 , TNF- e IL-6), excitotoxicidade (GFAP, TBARS) e neuroplasticidade (BDNF) em um modelo farmacológico de mania induzido pelo tratamento agudo e crônico com D – anfetamina (AMPH) (2mg/kg). Demonstramos na atividade locomotora uma aparente falta de capacidade de resposta à AMPH por animais com P2X7R bloqueado ou ausente (P2X7R-/-). Da mesma forma, observamos que o P2X7R participa do aumento do ambiente próinflamatório e excitotóxico induzido pela AMPH, demonstrado pelo papel de reversão do bloqueio do P2X7R ou pela perda de resposta dos animais P2X7R-/-. Em conclusão, nossos resultados suportam a hipótese de que o P2X7R possui um papel na patofisiologia do TB, como sugerido pelo nosso modelo animal, principalmente por mediar a neuroinflamação e a excitotoxicidade e finalmente levando a mudanças comportamentais. Assim, acreditamos que o P2X7R possui potencial para se tornar um alvo terapêutico do TB. Baseado neste cenário, estudos mais detalhados do papel do P2X7R no TB são necessários, especialmente devido à sua capacidade de agir sobre a microglia e modular a neuroinflamação e a excitotoxicidade. / The purinergic system has been increasingly implicated in medical conditions, among them bipolar disorder (BD). Up to date, no study has been conducted in order to clarify the possible contribution of the P2X7 receptor (P2X7R) to BD pathophysiology or even to review the previously data about this subject. In the presents works, we aim firstly to explore the association between the receptor and the disorder and identify the mechanisms by which the P2X7R plays a role in BD and lastly to review the literature focused on purinergic signaling in BD, with an emphasis on ATP and adenosine signaling, highlighting the potential role of P2X7R in modulating inflammation and microglia activation in bipolar patients. For the first aim, we analyzed the P2X7R modulatory effect (BzATP, BBG and A438079) on behavior (locomotor activity) and on markers of neuroinflammation (IL-1 , TNF- and IL-6), excitotoxicity (GFAP, TBARS), and neuroplasticity (BDNF) in a pharmacological model of mania induced by acute and chronic D-amphetamine (AMPH) treatment (2 mg/kg). We demonstrate in the locomotor activity an apparent lack of responsiveness to AMPH by animals with blocked or absent P2X7R (P2X7R-/-). Likewise, we observed that the P2X7R participates in the AMPH-induced increase of proinflammatory and excitotoxicity environment, demonstrated by the reversal role of P2X7R blockage or the lack of response by P2X7R-/- mice. In conclusion, our results provide support for the hypothesis that P2X7R plays a role in BD pathophysiology, as suggested by our animal model, mainly by mediating neuroinflammation and excitotoxicity and ultimately leading to behavioral changes. Thus, we believe that P2X7R has the potential to become a therapeutic target of BD. Based on this scenario, a more detailed study of the role of P2X7R in BD is warranted, especially due to its ability to act on microglia and modulate neuroinflammation and excitotoxicity.

O sistema purinérgico tem sido amplamente implicado em condições médicas, entre elas, o transtorno bipolar (TB). Até onde sabemos, nenhum estudo foi realizado com o objetivo de esclarecer a possível contribuição do receptor P2X7 (P2X7R) na patofisiologia do TB ou mesmo com o objetivo de revisar o que já fora previamente publicado sobre este assunto. Nos presentes trabalhos, nosso objetivo foi em primeiro lugar explorar a relação entre o receptor e esse transtorno e identificar os mecanismos pelos quais o P2X7R desempenha um papel no TB e por fim revisar a literatura, focando na sinalização purinérgica no TB, com ênfase na sinalização por ATP e adenosina, destacando o potencial papel do P2X7R na modulação da inflamação e ativação microglial em pacientes bipolares. Para o primeiro objetivo, analisamos o efeito de moduladores do P2X7R (BzATP, BBG e A438079) sobre o comportamento (atividade locomotora) e sobre marcadores de neuroinflamação (IL-1 , TNF- e IL-6), excitotoxicidade (GFAP, TBARS) e neuroplasticidade (BDNF) em um modelo farmacológico de mania induzido pelo tratamento agudo e crônico com D – anfetamina (AMPH) (2mg/kg). Demonstramos na atividade locomotora uma aparente falta de capacidade de resposta à AMPH por animais com P2X7R bloqueado ou ausente (P2X7R-/-). Da mesma forma, observamos que o P2X7R participa do aumento do ambiente próinflamatório e excitotóxico induzido pela AMPH, demonstrado pelo papel de reversão do bloqueio do P2X7R ou pela perda de resposta dos animais P2X7R-/-. Em conclusão, nossos resultados suportam a hipótese de que o P2X7R possui um papel na patofisiologia do TB, como sugerido pelo nosso modelo animal, principalmente por mediar a neuroinflamação e a excitotoxicidade e finalmente levando a mudanças comportamentais. Assim, acreditamos que o P2X7R possui potencial para se tornar um alvo terapêutico do TB. Baseado neste cenário, estudos mais detalhados do papel do P2X7R no TB são necessários, especialmente devido à sua capacidade de agir sobre a microglia e modular a neuroinflamação e a excitotoxicidade. / The purinergic system has been increasingly implicated in medical conditions, among them bipolar disorder (BD). Up to date, no study has been conducted in order to clarify the possible contribution of the P2X7 receptor (P2X7R) to BD pathophysiology or even to review the previously data about this subject. In the presents works, we aim firstly to explore the association between the receptor and the disorder and identify the mechanisms by which the P2X7R plays a role in BD and lastly to review the literature focused on purinergic signaling in BD, with an emphasis on ATP and adenosine signaling, highlighting the potential role of P2X7R in modulating inflammation and microglia activation in bipolar patients. For the first aim, we analyzed the P2X7R modulatory effect (BzATP, BBG and A438079) on behavior (locomotor activity) and on markers of neuroinflammation (IL-1 , TNF- and IL-6), excitotoxicity (GFAP, TBARS), and neuroplasticity (BDNF) in a pharmacological model of mania induced by acute and chronic D-amphetamine (AMPH) treatment (2 mg/kg). We demonstrate in the locomotor activity an apparent lack of responsiveness to AMPH by animals with blocked or absent P2X7R (P2X7R-/-). Likewise, we observed that the P2X7R participates in the AMPH-induced increase of proinflammatory and excitotoxicity environment, demonstrated by the reversal role of P2X7R blockage or the lack of response by P2X7R-/- mice. In conclusion, our results provide support for the hypothesis that P2X7R plays a role in BD pathophysiology, as suggested by our animal model, mainly by mediating neuroinflammation and excitotoxicity and ultimately leading to behavioral changes. Thus, we believe that P2X7R has the potential to become a therapeutic target of BD. Based on this scenario, a more detailed study of the role of P2X7R in BD is warranted, especially due to its ability to act on microglia and modulate neuroinflammation and excitotoxicity.

To establish comprehensive pharmacology of P2X₇ receptors, membrane current recording, intracellular calcium transient recording and ethidium bromide uptake were carried out to examine several selective (A-740003, A- 438079) and non-selective (suramin) P2X₇ antagonists across mammalian P2X₇ receptors (human, mouse and rat). These P2X₇ receptors demonstrated species-dependent sensitivities to antagonists. In each species, A-740003 revealed variant IC50 values with different assays, indicating the assay- dependent pharmacology of P2X₇ receptors. Conventionally, pharmacology can be used to define a native current but not in the case of the human breast cancer cell line, Hs578T. It is found that P2X₇ was expressed at both mRNA and protein level. The ATP-evoked currents recorded from Hs578T cells were P2X₇-like with distinctive electrophysiological features. But the pharmacology profile of the currents did not fit with P2X₇ receptor. Further experiments are needed to either include or exclude the existence of functional P2X₇ receptors in Hs578T. Transmembrane domain 2 (TM2) is known as the pore-forming region for P2X receptors. TM2 of P2X₇ receptor was investigated with cysteine substitution scanning. The predicted α-helix structure of the TM2 segment was in good agreement with the results from the substituted cysteine accessibility method (SCAM). Thr336, Ser339, Tyr343, Phe344 and Thr348 were found important for both channel dilation and aqueous pore formation. Ser339 was further studied. Various substitutions at Ser339 were explored. The results suggest that the polarity of the side chain at Ser339 is essential for the channel dilation. Furthermore, disulfide bond formation was identified between S339C in the trimeric receptor, implying that the side chains of Ser339 might turn very close to each other during the channel opening and dilation.

The cornea forms the anterior surface of the eye and is responsible for most of the eye’s refractive power. Injury to the outermost layer of the cornea, a non-keratinized stratified squamous epithelium, triggers a transient rise in intracellular calcium concentration that propagates radially from the wound. This calcium mobilization is initiated by the binding of nucleotides such as adenosine triphosphate (ATP), which are released from cells ruptured by the injury, to purinergic receptors (purinoreceptors) on undamaged cells near the wound. Downstream effects of this injury-induced "calcium wave" are generally thought to include the activation of signaling pathways that promote wound healing. However, the specific contributions of individual purinergic receptors to the overall wound response have in most cases not been well characterized. Purinoreceptors are classified into two broad categories: the P2Y class of G protein-coupled receptors, which act through second messengers to release calcium into the cytosol from the endoplasmic reticulum, and the P2X class of ligand-gated ionotropic receptors, which release calcium into the cytosol from the extracellular environment. Previously, our lab established the importance of the P2Y2 receptor to corneal epithelial wound healing by showing that P2Y2 activation makes a substantial contribution to the overall wound-induced calcium response, particularly in cells back from the leading edge, and promotes cell migration after injury. P2Y2 activation was also found to promote the phosphorylation of proteins involved in focal adhesions, which are multi-protein complexes that facilitate cell migration by transmitting the forces generated by the actin cytoskeleton to the extracellular environment. More recently, our lab has begun to demonstrate that P2X7 may play an equally important, yet distinct and perhaps complementary role in corneal epithelial wound healing. For instance, P2X7 was found to strongly influence the intensity of the injury-induced calcium response in cells immediately adjacent to the wound, and treatment with the P2X7 inhibitor oxidized ATP (oxATP) was shown to impair migration after injury both in vitro and in ex vivo rat corneas. Additionally, immunofluorescence of cells fixed eight hours after injury revealed an altered actin cytoskeletal architecture and localization of the focal adhesion proteins talin and vinculin in oxATP-treated cells compared to control cells. The goal of the present study was to further characterize P2X7’s role in the overall response to injury by using live cell imaging to examine actin cytoskeletal rearrangements and focal adhesion dynamics after injury under both control conditions and conditions of P2X7 inhibition. Human corneal limbal epithelial (HCLE) cells were transduced to express either actin or talin tagged with green fluorescent protein (GFP), grown into confluent monolayers, and scratch wounded in the presence or absence of oxATP. Cells at the leading edge of the wound were imaged using confocal microscopy every 10 minutes for 4 hours beginning 0.5 hours after injury. Analysis of the resulting actin-GFP movies revealed trends toward delayed extension of filopodia in oxATP-treated cells relative to control cells, as well as complex changes in the number of filopodia per cell over time. Additionally, while both groups formed lamella containing thick actin bundles that were oriented perpendicularly to the direction of migration, in oxATP-treated cells the formation of these structures was delayed. Furthermore, in oxATP-treated cells these actin bundles tended to persist once formed. This was in contrast to control cells, in which they tended to turn over to be replaced by thinner and shorter actin bundles that were oriented more obliquely relative to the direction of migration. Finally, analysis of talin-GFP movies demonstrated that focal adhesion lifespan was extended in oxATP-treated cells compared to control cells. Focal adhesions in oxATP-treated cells also exhibited a greater propensity to merge together or split apart, further suggesting impaired focal adhesion turnover. Overall, these findings suggest that P2X7 plays a critical role in promoting migration after corneal epithelial injury by coordinating rapid rearrangements of the actin cytoskeleton and turnover of focal adhesions at the leading edge.

Satellite glial cells (SGCs) surround the cell bodies of neurons of the peripheral nervous system, including those of the sensory ganglia. Their close apposition to the neuronal soma allows for bi-directional communication between neurons and SGCs, which are thought to regulate neuronal activity. After nerve injury, SGCs in the dorsal root ganglia contribute to neuropathic pain. Although the mechanisms are not fully understood, SGCs show increased coupling via gap junctions, and communicate with the neuron via bi-directional purinergic signaling after nerve injury. The increased coupling between SGCs and neurons may have implications for chronic pain following peripheral nerve injury. In vivo studies suggest that injury through the administration of capsaicin to the sensory nerve endings causes SGCs to be activated and proliferate. We have shown that capsaicin treatment in an in vitro co-culture of sensory neurons and SGCs increased the expression of the proliferation marker, Ki-67 in the glia. Here, we examine whether purinergic signaling plays a role in the promotion of SGC proliferation.

A Doença de Parkinson é uma doença altamente incapacitante e de grande prevalência. Pouco se sabe sobre sua etiologia e os tratamentos atuais consistem na diminuição dos sintomas, uma vez que ainda não foi encontrada uma maneira de reverter o déficit de neurônios dopaminérgicos observados nos pacientes acometidos. Sabe-se que os receptores purinérgicos são encontrados por todo o sistema nervoso central, não só no indivíduo adulto como também em diferentes estágios do desenvolvimento embrionário e estão envolvidos com proliferação e diferenciação celular. Este trabalho estudou a participação dos receptores purinérgicos em modelo animal de doença de Parkinson por lesão dos neurônios dopaminérgicos da via nigroestriatal com 6-OH dopamina (6-OHDA). Realizamos a análise do perfil de expressão gênica dos diferentes receptores após a lesão e subsequente modulação. Observamos expressão gênica alterada dos receptores P2X7 e P2Y6 até 5 semanas após a lesão. O uso do antagonista do receptor P2X7 Brilliant Blue G (BBG) induziu a regeneração da via nigroestriatal e o uso do antagonista do receptor P2Y6 MRS2578 preveniu a morte dos neurônios. Como esses efeitos foram acompanhados pela inativação de células microgliais, supõe-se que o controle do microambiente neuroinflamatório causado pela injeção de 6-OHDA seja a principal causa do efeito antiparkinsoniano observado pelo tratamento com BBG e MRS2578. Além disso, o transplante celular com células precursoras neuraisnão foi capaz de reverter o comportamento hemiparkinsoniano dos animais lesionados. Apesar do uso concomitante com BBG reduzir o comportamento, parece que esse efeito deve-se ao BBG per se, uma vez que o tratamento somente com o antagonista de P2X7 foi mais eficaz. De maneira geral, a modulação da atividade dos receptores purinérgicos se mostrou uma ferramenta promissora na pesquisa de cura e compreensão das bases moleculares da Doença de Parkinson / Parkinson\'s disease is a highly disabling and prevalent disease. Little is known about its etiology and the current treatments consist in the reduction of the symptoms, since there is no known method to reverse the dopaminergic neurons deficit observed in patients. Purinergic receptors are found throughout the central nervous system, not only in the adult individual but also at different stages of embryonic development, and are involved in proliferation and differentiation. This work investigated the role of purinergic receptors in the animal model of Parkinson\'s disease induced by 6-OH dopamine (6-OHDA) injection and consequent death of dopaminergic neurons of the nigrostriatal pathway. Patterns of purinergic receptors gene expression after the lesion and subsequent modulation were analyzed. We observed altered gene expression of P2X7 and P2Y6 receptors within 5 weeks of injury. The use of the P2X7 receptor antagonist Brilliant Blue G (BBG) induced the regeneration of the nigrostriatal pathway and treatment with P2Y6 receptor antagonist MRS2578 prevented the death of the neurons. Since these effects were accompanied by the inactivation of microglial cells, it is assumed that the control of neuroinflammatory milieu caused by the 6-OHDA injection is the main cause of the antiparkinsonian effect observed by the treatment with BBG and MRS2578. In addition, transplantation with neural precursor cells was not able to reverse the hemiparkinsonian behavior of injured animals. Although concomitant use with BBG improved cell engraftment, it appears that this effect is due to BBG per se, since treatment with only this P2X7receptor antagonist was more effective. In general, modulation of purinergic receptor activity showed to be a promising tool in the research of cure and understanding of the molecular bases of Parkinson\'s Disease.

A tuberculose (TB) é uma doença infecciosa causada por bactérias do gênero Mycobacterium que acomete principalmente o sistema respiratório. A cepa Beijing 1471 (M. tuberculosis) induz uma resposta altamente pró-inflamatória, enquanto que a cepa MP287/03 (M. bovis) induz uma fraca resposta inflamatória. O receptor P2X7 (P2X7R) é um sensor de ATP extracelular, uma molécula associada ao dano que é liberada a partir de células necróticas. A TB induzida por ambas as cepas hipervirulentas Beijing 1471 e MP287/03 é atenuada em camundongos deficientes do P2X7R. Portanto o objetivo do nosso trabalho foi avaliar o papel do P2X7R na resposta imune da TB induzida por essas cepas hipervirulentas. Nós percebemos que apesar das diferenças na capacidade imunomodulatória induzida pelas cepas Beijing 1471 e MP287/03, o P2X7R exerce um papel importante na severidade da doença induzida por ambas as cepas, e que a ausência desse receptor foi capaz de restabelecer a resposta imune pulmonar a perfis semelhantes ao induzido pela cepa de menor virulência H37Rv de M. tuberculosis. / Tuberculosis (TB) is infectious diseases caused by Mycobacterium tuberculosis (Mtb) that mainly affect the respiratory system. Beijing 1471 strain (Mtb) induce a strong pro-inflammatory response, while MP287/03 strain (M. bovis) induce a weak pro-inflammatory response. The P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine. TB caused by both hypervirulent strains Beijing 1471 and MP287/03 is attenuated in P2X7R deficient mice (P2X7RKO). Therefore, our aim was to investigate the role of P2X7R in imune response of TB induced by MP287/03 and Beijing 1471 strains. We has note that despite Beijing 1471 and MP287/03 strains have opposite immunomodulatory properties, the P2X7R have an important role in modulating the immune response induced by both strains. Thus, the lung immune responses induced by both hypervirulent infections in the absence of this receptor were similar to that induced by less virulent H37Rv Mtb mycobacteria.